Abstract
We propose a simple label-free electrochemical biosensor for monitoring protein kinase activity and inhibition using a peptide-modified electrode. The biosensor employs cys-kemptide (CLRRASLG) as a substrate peptide which was immobilized on the surface of a gold electrode via the self-assembly of the thiol terminals in cysteine (C) residues. The interaction between protein kinase A (PKA) and adenosine 5′-triphosphate (ATP) on the cys-kemptide immobilized electrode can cause the transfer of ATP terminal phosphates to the peptide substrates at serine (S) residues, which alters the surface charge of the electrode, thus enabling monitoring of the PKA activity via measuring the interfacial electron transfer resistance with electrochemical impedance spectroscopy. The proposed sensor showed reliable, sensitive, and selective detection of PKA activity with a wide dynamic range of 0.1–100 U/mL and a detection limit of 56 mU/mL. The sensor also exhibited high selectivity, rendering it possible to screen PKA inhibitors. Moreover, the sensor can be employed to evaluate the activity and inhibition of PKA in real samples.
Highlights
Protein phosphorylation by protein kinase is one of the most common and important post-translational modifications, representing the basis of numerous cellular processes, as well as many pathological conditions [1]
The negatively charged electrode surface. This result verifies the sensing principle of the proposed kemptide/AuE biosensor which can be employed for the label-free electrochemical detection of protein kinase A (PKA) activity by cyclic voltammetry (CV) and electrochemical impedanceimpedspecance spectroscopy (EIS) experiments
We have proposed a facile and label-free assay to detect PKA activity and inhibition using a peptide electrode-based electrochemical biosensor
Summary
Protein phosphorylation by protein kinase is one of the most common and important post-translational modifications, representing the basis of numerous cellular processes, as well as many pathological conditions [1]. For drug development and disease diagnosis of such pathological conditions, an effective monitoring system of protein kinase activity and inhibition is crucial to ensure reliable, sensitive, and selective detection. Alternative assays for protein kinase activity have been developed based on many different techniques, including colorimetry [6], mass spectrometry [7], fluorometry [8], flow cytometry [9], resonance light scattering [10], and electrochemical luminescence method [11]. We report a label-free peptide-based electrochemical biosensor for monitorHerein, we report a label-free peptide-based electrochemical biosensor for monitoring the activity and inhibition of protein kinase A (PKA). MCP, 3-mercapto-1-propanol; AuE, Au the detection of protein kinase A (PKA) activity and inhibition.
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